AAV5 vector and uses thereof

ABSTRACT

The present invention provides an adeno-associated virus 5 (AAV5) virus and vectors and particles derived therefrom. In addition, the present invention provides methods of delivering a nucleic acid to a cell using the AAV5 vectors and particles.

This application is a continuation of international applicationPCT/US99/11958 filed on May 28, 1999, which claims priority to U.S.provisional application Ser. No. 60/087,029 filed on May 28, 1998. ThePCT/US99/11958 international application and the 60/087,029 provisionalpatent application are herein incorporated by this reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides adeno-associated virus 5 (AAV5) andvectors derived therefrom. Thus, the present invention relates to AAV5vectors for and methods of delivering nucleic acids to cells ofsubjects.

2. Background Art

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family (for review see 28). AAV is distinct from the othermembers of this family by its dependence upon a helper virus forreplication. In the absence of a helper virus, AAV has been shown tointegrate in a locus specific manner into the q arm of chromosome 19(21). The approximately 5 kb genome of AAV consists of one segment ofsingle stranded DNA of either plus or minus polarity. Physically, theparvovirus virion is non-enveloped and its icosohedral capsid isapproximately 20–25 nm in diameter.

To date 8 serologically distinct AAVs have been identified and 6 havebeen isolated from humans or primates and are referred to as AAV types1–6 (1). The most extensively studied of these isolates is AAV type 2(AAV2). The genome of AAV2 is 4680 nucleotides in length and containstwo open reading frames (ORFs), the right ORF and the left ORF. The leftORF encodes the non-structural Rep proteins, Rep40, Rep52, Rep68 andRep78, which are involved in regulation of replication and transcriptionin addition to the production of single-stranded progeny genomes (5–8,11, 12, 15, 17, 19, 21–23, 25, 34, 37–40). Furthermore, two of the Repproteins have been associated with the preferential integration of AAVgenomes into a region of the q arm of human chromosome 19. Rep68/78 havealso been shown to possess NTP binding activity as well as DNA and RNAhelicase activities. The Rep proteins possess a nuclear localizationsignal as well as several potential phosphorylation sites. Mutation ofone of these kinase sites resulted in a loss of replication activity.

The ends of the genome are short inverted terminal repeats which havethe potential to fold into T-shaped hairpin structures that serve as theorigin of viral DNA replication. Within the ITR region two elements havebeen described which are central to the function of the ITR, a GAGCrepeat motif and the terminal resolution site (TRS). The repeat motifhas been shown to bind Rep when the ITR is in either a linear or hairpinconformation (7, 8, 26).

This binding serves to position Rep68/78 for cleavage at the TRS whichoccurs in a site- and strand-specific manner. In addition to their rolein replication, these two elements appear to be central to viralintegration. Contained within the chromosome 19 integration locus is aRep binding site with an adjacent TRS. These elements have been shown tobe functional and necessary for locus specific integration.

The AAV2 virion is a non-enveloped, icosohedral particle approximately20–25 nm in diameter. The capsid is composed of three related proteinsreferred to as VP 1,2 and 3 which are encoded by the right ORF. Theseproteins are found in a ratio of 1:1:10 respectively. The capsidproteins differ from each other by the use of alternative splicing andan unusual start codon. Deletion analysis of has shown that removal oralteration of AAV2 VP1 which is translated from an alternatively splicedmessage results in a reduced yield of infections particles (15, 16, 38).Mutations within the VP3 coding region result in the failure to produceany single-stranded progeny DNA or infectious particles (15, 16, 38).

The following features of the characterized AAVs have made themattractive vectors for gene transfer (16). AAV vectors have been shownin vitro to stably integrate into the cellular genome; possess a broadhost range; transduce both dividing and non dividing cells in vitro andin vivo (13, 20, 30, 32) and maintain high levels of expression of thetransduced genes (41). Viral particles are heat stable, resistant tosolvents, detergents, changes in pH, temperature, and can beconcentrated on CsCl gradients (1,2). Integration of AAV provirus is notassociated with any long term negative effects on cell growth ordifferentiation (3,42). The ITRs have been shown to be the only ciselements required for replication, packaging and integration (35) andmay contain some promoter activities (14).

AAV2 was originally thought to infect primate and non-primate cell typesprovided the appropriate helper virus was present. However, theinability of AAV2 to infect certain cell types is now known to be due tothe particular cellular tropism exhibited by the AAV2 virus. Recent workhas shown that some cell lines are transduced very poorly by AAV2 (30).Binding studies have indicated that heparin sulfate proteoglycans arenecessary for high efficiency transduction with AAV2. AAV5 is a uniquemember of the parvovirus family. The present DNA hybridization dataindicate a low level of homology with the published AAV1–4 sequences(31). The present invention shows that, unlike AAV2, AAV5 transductionis not effected by heparin as AAV2 is and therefore will not berestricted to the same cell types as AAV2.

The present invention provides a vector comprising the AAV5 virus or avector comprising subparts of the virus, as well as AAV5 viralparticles. While AAV5 is similar to AAV2, the two viruses are foundherein to be physically and genetically distinct. These differencesendow AAV5 with some unique properties and advantages which better suitit as a vector for gene therapy. For example, one of the limitingfeatures of using AAV2 as a vector for gene therapy is production oflarge amounts of virus. Using standard production techniques, AAV5 isproduced at a 10–50 fold higher level compared to AAV2. Because of itsunique TRS site and rep proteins, AAV5 should also have a distinctintegration locus compared to AAV2.

Furthermore, as shown herein, AAV5 capsid protein, again surprisingly,is distinct from AAV2 capsid protein and exhibits different tissuetropism, thus making AAV5 capsid-containing particles suitable fortransducing cell types for which AAV2 is unsuited or less well-suited.AAV2 and AAV5 have been shown to be serologically distinct and thus, ina gene therapy application, AAV5, and AAV5-derived vectors, would allowfor transduction of a patient who already possess neutralizingantibodies to AAV2 either as a result of natural immunological defenseor from prior exposure to AAV2 vectors. Another advantage of AAV5 isthat AAV5 cannot be rescued by other serotypes. Only AAV5 can rescue theintegrated AAV5 genome and effect replication, thus avoiding unintendedreplication of AAV5 caused by other AAV serotypes. Thus, the presentinvention, by providing these new recombinant vectors and particlesbased on AAV5 provides a new and highly useful series of vectors.

SUMMARY OF THE INVENTION

The present invention provides a nucleic acid vector comprising a pairof adeno-associated virus 5 (AAV5) inverted terminal repeats and apromoter between the inverted terminal repeats.

The present invention further provides an AAV5 particle containing avector comprising a pair of AAV2 inverted terminal repeats.

Additionally, the instant invention provides an isolated nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO:1 (AAV5genome). Furthermore, the present invention provides an isolated nucleicacid consisting essentially of the nucleotide sequence set forth in SEQID NO:1 (AAV5 genome).

The present invention provides an isolated nucleic acid encoding an AAV5Rep protein, for example, the nucleic acid as set forth in SEQ ID NO:10.Additionally provided is an isolated full-length AAV5 Rep protein or aunique fragment thereof. Additionally provided is an isolated AAV5 Rep40 protein having the amino acid sequence set forth in SEQ ID NO:12, ora unique fragment thereof. Additionally provided is an isolated AAV5 Rep52 protein having the amino acid sequence set forth in SEQ ID NO:2, or aunique fragment thereof. Additionally provided is an isolated AAV5 Rep68 protein, having the amino acid sequence set forth in SEQ ID NO:14 ora unique fragment thereof. Additionally provided is an isolated AAV5 Rep78 protein having the amino acid sequence set forth in SEQ ID NO:3, or aunique fragment thereof. The sequences for these proteins are providedbelow in the Sequence Listing and elsewhere in the application where theproteins are described.

The present invention further provides an isolated AAV5 capsid protein,VP 1, having the amino acid sequence set forth in SEQ ID NO:4, or aunique fragment thereof. Additionally provided is an isolated AAV5capsid protein, VP2, having the amino acid sequence set forth in SEQ IDNO:5, or a unique fragment thereof. Also provided is an isolated AAV5capsid protein, VP3, having the amino acid sequence set forth in SEQ IDNO:6, or a unique fragment thereof.

The present invention additionally provides an isolated nucleic acidencoding AAV5 capsid protein, for example, the nucleic acid set forth inSEQ ID NO:7, or a unique fragment thereof.

The present invention further provides an AAV5 particle comprising acapsid protein consisting essentially of the amino acid sequence setforth in SEQ ID NO:4, or a unique fragment thereof.

Additionally provided by the present invention is an isolated nucleicacid comprising an AAV5 p5 promoter having the nucleic acid sequence setforth in SEQ ID NO:18, or a unique fragment thereof.

The instant invention provides a method of screening a cell forinfectivity by AAV5 comprising contacting the cell with AAV5 anddetecting the presence of AAV5 in the cells.

The present invention further provides a method of delivering a nucleicacid to a cell comprising administering to the cell an AAV5 particlecontaining a vector comprising the nucleic acid inserted between a pairof AAV inverted terminal repeats, thereby delivering the nucleic acid tothe cell.

The present invention also provides a method of delivering a nucleicacid to a subject comprising administering to a cell from the subject anAAV5 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, and returning the cell to the subject,thereby delivering the nucleic acid to the subject.

The present invention also provides a method of delivering a nucleicacid to a cell in a subject comprising administering to the subject anAAV5 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, thereby delivering the nucleic acid to a cellin the subject.

The instant invention further provides a method of delivering a nucleicacid to a cell in a subject having antibodies to AAV2 comprisingadministering to the subject an AAV5 particle comprising the nucleicacid, thereby delivering the nucleic acid to a cell in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Heparin inhibition results. Cos cells were plated in 12well dishes at 5×10⁴ cells per well. Serial dilutions of AAV2 or AAV5produced and purified as previously described and supplemented with5×10⁵ particles of wt adenovirus were incubated for 1 hr at Rt in thepresence of 20 μg/ml heparin (sigma).

Following this incubation the virus was added to the cells in 400 μl ofmedia for 1 hr after which the media was removed, the cells rinsed andfresh media added. After 24 hrs the plates were stained for Bgalactivity.

FIG. 2 shows AAV2 and AAV5 vector and helper complementation.Recombinant AAV particles were produced as previously described using avariety of vector and helper plasmids as indicated the bottom of thegraph. The vector plasmids contained the Bgal gene with and RSV promoterand flanked by either AAV2 ITRs (2ITR) or AAV5 ITRs (5ITR). The helperplasmids tested contained either AAV2 Rep and cap genes (2repcap) AAV5rep and cap genes with or without an SV40 promoter (5repcapA and5repcapb respectively) only the AAV2 rep gene (2rep) in varying amounts(1) or (0.5) or an empty vector (pUC). The resulting AAV particles werethen titered on cos cells. AAV particles were only produced when thesame serotype of ITR and Rep were present.

FIG. 3 shows AAV2 and AAV5 tissue tropism. Transduction of a variety ofcell types indicated that AAV2 and AAV5 transduce cells with differentefficiencies.

Equal number of either AAV2 or AAV5 particles were used to transduce avariety of cell types and the number of bgal positive cells is reported.

FIG. 4 (Parts A–I) is a sequence comparison of the AAV2 genome (SEQ IDNO: 25) and the AAV5 genome (SEQ ID NO: 1).

FIG. 5 (Parts A–B) is a sequence comparison of the AAV2 VP1 capsidprotein (SEQ ID NO: 24) and the AAV5 VP1 capsid protein (SEQ ID NO: 4).

FIG. 6 (Parts A–B) is a sequence comparison of the AAV2 rep 78 protein(SEQ ID NO: 26) and the AAV5 rep 78 protein (SEQ ID NO: 3).

FIG. 7 shows the transduction of airway epithelial cells by AAV5.Primary airway epithelial cells were cultured and plated. Cells weretransducted with an equivalent number of rAAV2 or rAAV5 particlescontaining a nuclear localized β-gal transgene with 50 particles ofvirus/cell (MOI 50) and continued in culture for 10 days. β-gal activitywas determined and the relative transduction efficiency compared. AAV5transduced these cells 50-fold more efficiently than AAV2. This is thefirst time apical cells or cells exposed to the air have been shown tobe infected by a gene therapy agent.

FIG. 8 shows transduction of striated muscle by AAV5. Chicken myoblastswere cultured and plated. Cells were allowed to fuse and then transducedwith a similar number of particles of rAAV2 or rAAV5 containing anuclear localized β-gal transgene after 5 days in culture. The cellswere stained for β-gal activity and the relative transduction efficiencycompared. AAV5 transduced these cells approximately 16 fold moreefficiently than AAV2.

FIG. 9 shows transduction of rat brain explants by AAV5. Primaryneonatal rat brain explants were prepared. After 7 days in culture,cells were transduced with a similar number of particles of rAAV5containing a nuclear localized β-gal transgene. After 5 days in culture,the cells were stained for β-gal activity. Transduction was detected ina variety of cell types including astrocytes, neuronal cells and glialcells.

FIG. 10 shows transduction of human umbilical vein endothelial cells byAAV5. Human umbilical vein endothelial cells were cultured and plated.Cells were transduced with rAAV2 or rAAV5 containing a nuclear localizedβ-gal transgene with 10 particles of virus/cell (MOI 5) in minimal mediathen returned to complete media. After 24 hrs in culture, the cells werestained for β-gal activity and the relative transduction efficiencycompared. As shown in AAV5 transduced these cell 5–10 fold moreefficiently than AAV2.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification and in the claims, “a” can mean one ormore, depending upon the context in which it is used. The terms “having”and “comprising” are used interchangeably herein, and signify open endedmeaning.

The present application provides a recombinant adeno-associated virus 5(AAV5). This virus has one or more of the characteristics describedbelow. The compositions of the present invention do not includewild-type AAV5. The methods of the present invention can use eitherwild-type AAV5 or recombinant AAV5-based delivery.

The present invention provides novel AAV5 particles, recombinant AAV5vectors, recombinant AAV5 virions and novel AAV5 nucleic acids andpolypeptides. An AAV5 particle is a viral particle comprising an AAV5capsid protein. A recombinant AAV5 vector is a nucleic acid constructthat comprises at least one unique nucleic acid of AAV5. A recombinantAAV5 virion is a particle containing a recombinant AAV5 vector, wherinthe particle can be either an AAV5 particle as described herein or anon-AAV5 particle. Alternatively, the recombinant AAV5 virion is an AAV5particle containing a recombinant vector, wherein the vector can beeither an AAV5 vector as described herein or a non-AAV5 vector. Thesevectors, particles, virions, nucleic acids and polypeptides aredescribed below.

The present invention provides the nucleotide sequence of the AAV5genome and vectors and particles derived therefrom. Specifically, thepresent invention provides a nucleic acid vector comprising a pair ofAAV5 inverted terminal repeats (ITRs) and a promoter between theinverted terminal repeats. While the rep proteins of AAV2 and AAV5 willbind to either a type 2 ITR or a type 5 ITR, efficient genomereplication only occurs when type 2 Rep replicates a type 2 ITR and atype 5 Rep replicates a type 5 ITR. This specificity is the result of adifference in DNA cleavage specificity of the two Reps which isnecessary for replication. AAV5 Rep cleaves at CGGT^GTGA (SEQ ID NO: 21)and AAV2 Rep cleaves at CGGT^TGAG (SEQ ID NO: 22) (Chiorini et al.,1999. J. Virol. 73 (5) 4293–4298). Mapping of the AAV5 ITR terminalresolution site (TRS) identified this distinct cleavage site, CGGT^GTGA,which is absent from the ITRs of other AAV serotypes. Therefore, theminimum sequence necessary to distinguish AAV5 from AAV2 is the TRS sitewhere Rep cleaves in order to replicate the virus. Examples of the type5 ITRs are shown in SEQ ID NO: 19 and SEQ ID NO: 20, AAV5 ITR “flip” andAAV5 “flop”, respectively. Minor modifications in an ITR of eitherorientation are contemplated and are those that will not interfere withthe hairpin structure formed by the AAV5 ITR as described herein andknown in the art. Furthermore, to be considered within the term “AAV5ITR” the nucleotide sequence must retain one or more features describedherein that distinguish the AAV5 ITR from the ITRs of other serotypes,e.g. it must retain the Rep binding site described herein.

The D-region of the AAV5 ITR (SEQ ID NO: 23), a single stranded regionof the ITR, inboard of the TRS site, has been shown to bind a factorwhich depending on its phosphorylation state correlates with theconversion of the AAV from a single stranded genome to atranscriptionally active form that allows for expression of the viralDNA. This region is conserved between AAV2, 3, 4, and 6 but is divergentin AAV5. The D+ region is the reverse complement of the D-region.

The promoter can be any desired promoter, selected by knownconsiderations, such as the level of expression of a nucleic acidfunctionally linked to the promoter and the cell type in which thevector is to be used. That is, the promoter can be tissue/cell-specific.Promoters can be prokaryotic, eukaryotic, fungal, nuclear,mitochondrial, viral or plant promoters. Promoters can be exogenous orendogenous to the cell type being transduced by the vector. Promoterscan include, for example, bacterial promoters, known strong promoterssuch as SV40 or the inducible metallothionein promoter, or an AAVpromoter, such as an AAV p5 promoter. Additionally, chimeric regulatorypromoters for targeted gene expression can be utilized. Examples ofthese regulatory systems, which are known in the art, include thetetracycline based regulatory system which utilizes the tettransactivator protein (tTA), a chimeric protein containing the VP16activation domain fused to the tet repressor of Escherichia coli, theIPTG based regulatory system, the CID based regulatory system, and theEcdysone based regulatory system (44). Other promoters include promotersderived from actin genes, immunoglobulin genes, cytomegalovirus (CMV),adenovirus, bovine papilloma virus, adenoviral promoters, such as theadenoviral major late promoter, an inducible heat shock promoter,respiratory syncytial virus, Rous sarcomas virus (RSV), etc.,specifically, the promoter can be AAV2 p5 promoter or AAV5 p5 promoter.More specifically, the AAV5 p5 promoter can be about same location inSEQ ID NO: 1 as the AAV2 p5 promoter, in the corresponding AAV2published sequence. Additionally, the p5 promoter may be enhanced bynucleotides 1–130 of SEQ ID NO: 1. Furthermore, smaller fragments of p5promoter that retain promoter activity can readily be determined bystandard procedures including, for example, constructing a series ofdeletions in the p5 promoter, linking the deletion to a reporter gene,and determining whether the reporter gene is expressed, i.e.,transcribed and/or translated. The promoter can be the promoter of anyof the AAV serotypes, and can be the p19 promoter (SEQ ID NO: 16) or thep40 promoter set forth in the sequence listing as SEQ ID NO: 17.

It should be recognized that any errors in any of the nucleotidesequences disclosed herein can be corrected, for example, by using thehybridization procedure described below with various probes derived fromthe described sequences such that the coding sequence can be reisolatedand resequenced. Rapid screening for point mutations can also beachieved with the use of polymerase chain reaction-single strandconformation polymorphism (PCR-SSCP) (43). The corresponding amino acidsequence can then be corrected accordingly.

The AAV5-derived vector of the invention can further comprise aheterologous nucleic acid functionally linked to the promoter. By“heterologous nucleic acid” is meant that any heterologous or exogenousnucleic acid, i.e. not normally found in wild-type AAV5 can be insertedinto the vector for transfer into a cell, tissue or organism. By“functionally linked” is meant that the promoter can promote expressionof the heterologous nucleic acid, as is known in the art, and caninclude the appropriate orientation of the promoter relative to theheterologous nucleic acid. Furthermore, the heterologous nucleic acidpreferably has all appropriate sequences for expression of the nucleicacid. The nucleic acid can include, for example, expression controlsequences, such as an enhancer, and necessary information processingsites, such as ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

The heterologous nucleic acid can encode beneficial proteins orpolypeptides that replace missing or defective proteins required by thecell or subject into which the vector is transferred or can encode acytotoxic polypeptide that can be directed, e.g., to cancer cells orother cells whose death would be beneficial to the subject. Theheterologous nucleic acid can also encode antisense RNAs that call bindto, and thereby inactivate, mRNAs made by the subject that encodeharmful proteins. The heterologous nucleic acid can also encoderibozymes that can effect the sequence-specific inhibition of geneexpression by the cleavage of mRNAs. In one embodiment, antisensepolynucleotides can be produced from a heterologous expression cassettein an AAV5 vector construct where the expression cassette contains asequence that promotes cell-type specific expression (Wirak et al., EMBO10:289 (1991)). For general methods relating to antisensepolynucleotides, see Antisense RNA and DNA, D. A. Melton, Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).

Examples of heterologous nucleic acids which can be administered to acell or subject as part of the present AAV5 vector can include, but arenot limited to the following: nucleic acids encoding secretory andnonsecretory proteins, nucleic acids encoding therapeutic agents, suchas tumor necrosis factors (TNF), such as TNF-α; interferons, such asinterferon-α, interferon-P, and interferon-γ; interleukins, such asIL-1, IL-1β, and ILs-2 through-14; GM-CSF; adenosine deaminase; cellulargrowth factors, such as lymphokines; soluble CD4; Factor VIII; FactorIX; T-cell receptors; LDL receptor; ApoE; ApoC; alpha-1 antitrypsin;ornithine transcarbamylase (OTC); cystic fibrosis transmembrane receptor(CFTR); insulin; Fc receptors for antigen binding domains of antibodies,such as immunoglobulins; anit-HIV decoy tar elements; and antisensesequences which inhibit viral replication, such as antisense sequenceswhich inhibit replication of hepatitis B or hepatitis non-A, non-Bvirus. The nucleic acid is chosen considering several factors, includingthe cell to be transfected. Where the target cell is a blood cell, forexample, particularly useful nucleic acids to use are those which allowthe blood cells to exert a therapeutic effect, such as a gene encoding aclotting factor for use in treatment of hemophilia. Another target cellis the lung airway cell, which can be used to administer nucleic acids,such as those coding for the cystic fibrosis transmembrane receptor,which could provide a gene therapeutic treatment for cystic fibrosis.Other target cells include muscle cells where useful nucleic acids, suchas those encoding cytokines and growth factors, can be transduced andthe protein the nucleic acid encodes can be expressed and secreted toexert its effects on other cells, tissues and organs, such as the liver.Furthermore, the nucleic acid can encode more than one gene product,limited only, if the nucleic acid is to be packaged in a capsid, by thesize of nucleic acid that can be packaged.

Furthermore, suitable nucleic acids can include those that, whentransferred into a primary cell, such as a blood cell, cause thetransferred cell to target a site in the body where that cell's presencewould be beneficial. For example, blood cells such as TIL cells can bemodified, such as by transfer into the cell of a Fab portion of amonoclonal antibody, to recognize a selected antigen. Another examplewould be to introduce a nucleic acid that would target a therapeuticblood cell to tumor cells. Nucleic acids useful in treating cancer cellsinclude those encoding chemotactic factors which cause an inflammatoryresponse at a specific site, thereby having a therapeutic effect.

Cells, particularly blood cells, muscle cells, airway epithelial cells,brain cells and endothelial cells having such nucleic acids transferredinto them can be useful in a variety of diseases, syndromes andconditions. For example, suitable nucleic acids include nucleic acidsencoding soluble CD4, used in the treatment of AIDS and α-antitrypsin,used in the treatment of emphysema caused by α-antitrypsin deficiency.Other diseases, syndromes and conditions in which such cells can beuseful include, for example, adenosine deaminase deficiency, sickle celldeficiency, brain disorders such as Alzheimer's disease, thalassemia,hemophilia, diabetes, phenylketonuria, growth disorders and heartdiseases, such as those caused by alterations in cholesterol metabolism,and defects of the immune system.

As another example, hepatocytes can be transfected with the presentvectors having useful nucleic acids to treat liver disease. For example,a nucleic acid encoding OTC can be used to transfect hepatocytes (exvivo and returned to the liver or in vivo) to treat congenitalhyperammonemia, caused by an inherited deficiency in OTC. Anotherexample is to use a nucleic acid encoding LDL to target hepatocytes exvivo or in vivo to treat inherited LDL receptor deficiency. Suchtransfected hepatocytes can also be used to treat acquired infectiousdiseases, such as diseases resulting from a viral infection. Forexample, transduced hepatocyte precursors can be used to treat viralhepatitis, such as hepatitis B and non-A, non-B hepatitis, for exampleby transducing the hepatocyte precursor with a nucleic acid encoding anantisense RNA that inhibits viral replication. Another example includestransferring a vector of the present invention having a nucleic acidencoding a protein, such as α-interferon, which can confer resistance tothe hepatitis virus.

For a procedure using transfected hepatocytes or hepatocyte precursors,hepatocyte precursors having a vector of the present inventiontransferred in can be grown in tissue culture, removed from the tissueculture vessel, and introduced to the body, such as by a surgicalmethod. In this example, the tissue would be placed directly into theliver, or into the body cavity in proximity to the liver, as in atransplant or graft. Alternatively, the cells can simply be directlyinjected into the liver, into the portal circulatory system, or into thespleen, from which the cells can be transported to the liver via thecirculatory system. Furthermore, the cells can be attached to a support,such as microcarrier beads, which can then be introduced, such as byinjection, into the peritoneal cavity. Once the cells are in the liver,by whatever means, the cells can then express the nucleic acid and/ordifferentiate into mature hepatocytes which can express the nucleicacid.

The AAV5-derived vector can include any normally occurring AAV5sequences in addition to an ITR and promoter. Examples of vectorconstructs are provided below.

The present vector or AAV5 particle or recombinant AAV5 virion canutilize any unique fragment of these present AAV5 nucleic acids,including the AAV5 nucleic acids set forth in SEQ ID NOS: 1 and 7–11,13, 15, 16, 17, and 18. To be unique, the fragment must be of sufficientsize to distinguish it from other known sequences, most readilydetermined by comparing any nucleic acid fragment to the nucleotidesequences of nucleic acids in computer databases, such as GenBank. Suchcomparative searches are standard in the art. Typically, a uniquefragment useful as a primer or probe will be at least about 8 or 10,preferable at least 20 or 25 nucleotides in length, depending upon thespecific nucleotide content of the sequence. Additionally, fragments canbe, for example, at least about 30, 40, 50, 75, 100, 200 or 500nucleotides in length and can encode polypeptides or be probes. Thenucleic acid can be single or double stranded, depending upon thepurpose for which it is intended. Where desired, the nucleic acid can beRNA.

The present invention further provides an AAV5 capsid protein to containthe vector. In particular, the present invention provides not only apolypeptide comprising all three AAV5 coat proteins, i.e., VP1, VP2 andVP3, but also a polypeptide comprising each AAV5 coat proteinindividually, SEQ ID NOS: 4, 5, and 6, respectively. Thus an AAV5particle comprising an AAV5 capsid protein comprises at least one AAV5coat protein VP 1, VP2 or VP3. An AAV5 particle comprising an AAV5capsid protein can be utilized to deliver a nucleic acid vector to acell, tissue or subject. For example, the herein described AAV5 vectorscan be encapsidated in an AAV5 capsid-derived particle and utilized in agene delivery method. Furthermore, other viral nucleic acids can beencapsidated in the AAV5 particle and utilized in such delivery methods.For example, an AAV1, 2,3,4, or 6 vector (e.g. AAV1,2,3,4, or 6 ITR andnucleic acid of interest) can be encapsidated in an AAV5 particle andadministered. Furthermore, an AAV5 chimeric capsid incorporating bothAAV2 capsid and AAV5 capsid sequences can be generated, by standardcloning methods, selecting regions from the known sequences of eachprotein as desired. For example, particularly antigenic regions of theAAV2 capsid protein can be replaced with the corresponding region of theAAV5 capsid protein. In addition to chimeric capsids incorporating AAV2capsid sequences, chimeric capsids incorporating AAV1, 3, 4, or 6 andAAV5 capsid sequences can be generated, by standard cloning methods,selecting regions from the known sequences of each protein as desired.

The capsids can also be modified to alter their specific tropism bygenetically altering the capsid to encode a specific ligand to a cellsurface receptor. Alternatively, the capsid can be chemically modifiedby conjugating a ligand to a cell surface receptor. By genetically orchemically altering the capsids, the tropism can be modified to directAAV5 to a particular cell or population of cells. The capsids can alsobe altered immunologically by conjugating the capsid to an antibody thatrecognizes a specific protein on the target cell or population of cells.

The capsids can also be assembled into empty particles by expression inmammalian, bacterial, fungal or insect cells. For example, AAV2particles are known to be made from VP3 and VP2 capsid proteins inbaculovirus. The same basic protocol can produce an empty AAV5 particlecomprising an AAV5 capsid protein.

The herein described recombinant AAV5 nucleic acid derived vector can beencapsidated in an AAV particle. In particular, it can be encapsidatedin an AAV1 particle, an AAV2 particle, an AAV3 particle, an AAV4particle, an AAV5 particle or an AAV6 particle, a portion of any ofthese capsids, or a chimeric capsid particle as described above, bystandard methods using the appropriate capsid proteins in theencapsidation process, as long as the nucleic acid vector fits withinthe size limitation of the particle utilized. The encapsidation processitself is standard in the art. The AAV5 replication machinery, i.e. therep initiator proteins and other functions required for replication, canbe utilized to produce the AAV5 genome that can be packaged in an AAV1,2, 3, 4, 5 or 6 capsid.

The recombinant AAV5 virion containing a vector can also be produced byrecombinant methods utilizing multiple plasmids. In one example, theAAV5 rep nucleic acid would be cloned into one plasmid, the AAV5 ITRnucleic acid would be cloned into another plasmid and the AAV1, 2, 3, 4,5 or 6 capsid nucleic acid would be cloned on another plasmid. Theseplasmids would then be introduced into cells. The cells that wereefficiently transduced by all three plasmids, would exhibit specificintegration as well as the ability to produce AAV5 recombinant virus.Additionally, two plasmids could be used where the AAV5 rep nucleic acidwould be cloned into one plasmid and the AAV5 ITR and AAV5 capsid wouldbe cloned into another plasmid. These plasmids would then be introducedinto cells. The cells that were efficiently transduced by both plasmids,would exhibit specific integration as well as the ability to produceAAV5 recombinant virus.

An AAV5 capsid polypeptide encoding the entire VP1, VP2, and VP3polypeptide can overall has greater than 56% homology to the polypeptidehaving the amino acid sequence encoded by nucleotides in SEQ ID NOS:7,8and 9, as shown in FIGS. 4 and 5. The capsid protein can have about 70%homology, about 75% homology, 80% homology, 85% homology, 90% homology,95% homology, 98% homology, 99% homology, or even 100% homology to theprotein having the amino acid sequence encoded by the nucleotides setforth in SEQ ID NOS:7, 8 or 9. The percent homology used to identifyproteins herein, can be based on a nucleotide-by-nucleotide comparisonor more preferable is based on a computerized algorithm as describedherein. Variations in the amino acid sequence of the AAV5 capsid proteinare contemplated herein, as long as the resulting particle comprising anAAV5 capsid protein remains antigenically or immunologically distinctfrom AAV 1, AAV2, AAV3, AAV4 or AAV6 capsid, as can be routinelydetermined by standard methods. Specifically, for example, ELISA andWestern blots can be used to determine whether a viral particle isantigenically or immunologically distinct from AAV2 or the otherserotypes. Furthermore, the AAV5 particle preferably retains tissuetropism distinction from AAV2, such as that exemplified in the examplesherein. An AAV5 chimeric particle comprising at least one AAV5 coatprotein may have a different tissue tropism from that of an AAV5particle consisting only of AAV5 coat proteins, but is still distinctfrom the tropism of an AAV2 particle.

The invention further provides a recombinant AAV5 virion, comprising anAAV5 particle containing, i.e., encapsidating, a vector comprising apair of AAV5 inverted terminal repeats. The recombinant vector canfurther comprise an AAV5 Rep-encoding nucleic acid. The vectorencapsidated in the particle can further comprise an exogenous nucleicacid inserted between the inverted terminal repeats. AAV5 Rep conferstargeted integration and efficient replication, thus production ofrecombinant AAV5, comprising AAV5 Rep, yields more particles thanproduction of recombinant AAV2. Since AAV5 is more efficient atreplicating and packaging its genome, the exogenous nucleic acidinserted, or in the AAV5 capsids of the present invention, between theinverted terminal repeats can be packaged in the AAV1, 2, 3, 4, or 6capsids to achieve the specific tissue tropism conferred by the capsidproteins.

The invention further contemplates chimeric recombinant ITRs thatcontains a rep binding site and a TRS site recognized by that Repprotein. By “Rep protein” is meant all four of the Rep proteins, Rep 40,Rep 78, Rep 52, Rep 68. Alternatively, “Rep protein” could be one ormore of the Rep proteins described herein. One example of a chimeric ITRwould consist of an AAV5 D region (SEQ ID NO: 23), an AAV5 TRS site (SEQID NO: 21), an AAV2 hairpin and an AAV2 binding site. Another examplewould be an AAV5 D region, an AAV5 TRS site, an AAV3 hairpin and an AAV3binding site. In these chimeric ITRs, the D region can be from AAV 1, 2,3, 4, 5 or 6. The hairpin can be derived from AAV 1,2 3, 4, 5, 6. Thebinding site can be derived from any of AAV1, 2, 3, 4, 5 or 6.Preferably, the D region and the TRS are from the same serotype.

The chimeric ITRs can be combined with AAV5 Rep protein and any of theAAV serotype capsids to obtain recombinant virion. For example,recombinant virion can be produced by an AAV5 D region, an AAV5 TRSsite, an AAV2 hairpin, an AAV2 binding site, AAV5 Rep protein and AAV1capsid. This recombinant virion would possess the cellular tropismconferred by the AAV 1 capsid protein and would possess the efficientreplication conferred by the AAV5 Rep.

Other examples of the ITR, Rep protein and Capsids that will producerecombinant virus are provided in the list below:

-   5ITR+5Rep+5Cap=virus-   5ITR+5Rep+1Cap=virus-   5ITR+5Rep+2Cap=virus-   5ITR+5Rep+3Cap=virus-   5ITR+5Rep+4Cap=virus-   5ITR+5Rep+6Cap=virus-   1ITR+1Rep+5Cap=virus-   2ITR+2Rep+5Cap=virus-   3ITR+3Rep+5Cap=virus-   4ITR+4Rep+5Cap=virus-   6ITR+6Rep+5Cap=virus

In any of the constructs described herein, inclusion of a promoter ispreferred. As used in the constructs herein, unless otherwise specified,Cap (capsid) refers to any of AAV5 VP1, AAV5 VP2, AAV5 VP3, combinationsthereof, functional fragments of any of VP1, VP2 or VP3, or chimericcapsids as described herein. The ITRs of the constructs describedherein, can be chimeric recombinant ITRs as described elsewhere in theapplication.

Conjugates of recombinant or wild-type AAV5 virions and nucleic acids orproteins can be used to deliver those molecules to a cell. For example,the purified AAV5 can be used as a vehicle for delivering DNA bound tothe exterior of the virus. Examples of this are to conjugate the DNA tothe virion by a bridge using poly-L-lysine or other charged molecule.Also contemplated are virosomes that contain AAV5 structural proteins(AAV5 capsid proteins), lipids such as DOTAP, and nucleic acids that arecomplexed via charge interaction to introduce DNA into cells.

Also provided by this invention are conjugates that utilize the AAV5capsid or a unique region of the AAV5 capsid protein (e.g. VP1, VP2 orVP3 or combinations thereof) to introduce DNA into cells. For example,the type 5 VP3 protein or fragment thereof, can be conjugated to a DNAon a plasmid that is conjugated to a lipid. Cells can be infected usingthe targeting ability of the VP3 capsid protein to achieve the desiredtissue tropism, specific to AAV5. Type 5 VP1 and VP2 proteins can alsobe utilized to introduce DNA or other molecules into cells. By furtherincorporating the Rep protein and the AAV TRS into the DNA-containingconjugate, cells can be transduced and targeted integration can beachieved. For example, if AAV5 specific targeted integration is desired,a conjugate composed of the AAV5 VP3 capsid, AAV5 rep or a fragment ofAAV5 rep, AAV5 TRS, the rep binding site, the heterologous DNA ofinterest, and a lipid, can be utilized to achieve AAV5 specific tropismand AAV5 specific targeted integration in the genome.

Further provided by this invention are chimeric viruses where AAV5 canbe combined with herpes virus, baculovirus or other viruses to achieve adesired tropism associated with another virus. For example, the AAV5ITRs could be inserted in the herpes virus and cells could be infected.Post-infection, the ITRs of AAV5 could be acted on by AAV5 rep providedin the system or in a separate vehicle to rescue AAV5 from the genome.Therefore, the cellular tropism of the herpes simplex virus can becombined with AAV5 rep mediated targeted integration. Other viruses thatcould be utilized to construct chimeric viruses include, lentivirus,retrovirus, pseudotyped retroviral vectors, and adenoviral vectors.

The present invention further provides isolated nucleic acids of AAV5.For example, provided is an isolated nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO:1 (AAV5 genome). This nucleicacid, or portions thereof, can be inserted into vectors, such asplasmids, yeast artificial chromosomes, or other viral vector(particle), if desired, by standard cloning methods. The presentinvention also provides an isolated nucleic acid consisting essentiallyof the nucleotide sequence set forth in SEQ ID NO:1. The nucleotides ofSEQ ID NO:1 can have minor modifications and still be contemplated bythe present invention. For example, modifications that do not alter theamino acid encoded by any given codon (such as by modification of thethird, “wobble,” position in a codon) can readily be made, and suchalterations are known in the art. Furthermore, modifications that causea resulting neutral (conserved) amino acid substitution of a similaramino acid can be made in a coding region of the genome. Additionally,modifications as described herein for the AAV5 components, such as theITRs, the p5 promoter, etc. are contemplated in this invention.Furthermore, modifications to regions of SEQ ID NO:1 other than in theITR, TRS Rep binding site and hairpin are likely to be tolerated withoutserious impact on the function of the nucleic acid as a recombinantvector.

As used herein, the term “isolated” refers to a nucleic acid separatedor significantly free from at least some of the other components of thenaturally occurring organism, for example, the cell structuralcomponents or viral components commonly found associated with nucleicacids in the environment of the virus and/or other nucleic acids. Theisolation of the native nucleic acids can be accomplished, for example,by techniques such as cell lysis followed by phenol plus chloroformextraction, followed by ethanol precipitation of the nucleic acids. Thenucleic acids of this invention can be isolated from cells according toany of many methods well known in the art.

As used herein, the term “nucleic acid” refers to single-or multiplestranded molecules which may be DNA or RNA, or any combination thereof,including modifications to those nucleic acids. The nucleic acid mayrepresent a coding strand or its complement, or any combination thereof.Nucleic acids may be identical in sequence to the sequences which arenaturally occurring for any of the novel genes discussed herein or mayinclude alternative codons which encode the same amino acid as thoseprovided herein, including that which is found in the naturallyoccurring sequence. These nucleic acids can also be modified from theirtypical structure. Such modifications include, but are not limited to,methylated nucleic acids, the substitution of a non-bridging oxygen onthe phosphate residue with either a sulfur (yielding phosphorothioatedeoxynucleotides), selenium (yielding phosphorselenoatedeoxynucleotides), or methyl groups (yielding methylphosphonatedeoxynucleotides).

The present invention additionally provides an isolated nucleic acidthat selectively hybridizes with any nucleic acid disclosed herein,including the entire AAV5 genome and any unique fragment thereof,including the Rep and capsid encoding sequences (e.g. SEQ ID NOS: 1, 7,8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 22 and 23). Specifically,the nucleic acid can selectively or specifically hybridize to anisolated nucleic acid consisting of the nucleotide sequence set forth inSEQ ID NO: 1 (AAV5 genome). The present invention further provides anisolated nucleic acid that selectively or specifically hybridizes withan isolated nucleic acid comprising the nucleotide sequence set forth inSEQ ID NO:1 (AAV5 genome). By “selectively hybridizes” as used herein ismeant a nucleic acid that hybridizes to one of the disclosed nucleicacids under sufficient stringency conditions without significanthybridization to a nucleic acid encoding an unrelated protein, andparticularly, without detectably hybridizing to nucleic acids of AAV2.Thus, a nucleic acid that selectively hybridizes with a nucleic acid ofthe present invention will not selectively hybridize under stringentconditions with a nucleic acid encoding a different protein or thecorresponding protein from a different serotype of the virus, and viceversa. A “specifically hybridizing” nucleic acid is one that hybridizesunder stringent conditions to only a nucleic acid found in AAV5.Therefore, nucleic acids for use, for example, as primers and probes todetect or amplify the target nucleic acids are contemplated herein.Nucleic acid fragments that selectively hybridize to any given nucleicacid can be used, e.g., as primers and or probes for furtherhybridization or for amplification methods (e.g., polymerase chainreaction (PCR), ligase chain reaction (LCR)). Additionally, for example,a primer or probe can be designed that selectively hybridizes with bothAAV5 and a gene of interest carried within the AAV5 vector (i.e., achimeric nucleic acid).

Stringency of hybridization is controlled by both temperature and saltconcentration of either or both of the hybridization and washing steps.Typically, the stringency of hybridization to achieve selectivehybridization involves hybridization in high ionic strength solution(6×SSC or 6×SSPE) at a temperature that is about 12–25° C. below theT_(m) (the melting temperature at which half of the molecules dissociatefrom their hybridization partners) followed by washing at a combinationof temperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the T_(m). The temperatureand salt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA—RNA hybridizations. The washingtemperatures can be used as described above to achieve selectivestringency, as is known in the art. (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987).A preferable stringent hybridization condition for a DNA:DNAhybridization can be at about 68° C. (in aqueous solution) in 6×SSC or6×SSPE followed by washing at 68° C. Stringency of hybridization andwashing, if desired, can be reduced accordingly as the degree ofcomplementarity desired is decreased, and further, depending upon theG-C or A-T richness of any area wherein variability is searched for.Likewise, stringency of hybridization and washing, if desired, can beincreased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

A nucleic acid that selectively hybridizes to any portion of the AAV5genome is contemplated herein. Therefore, a nucleic acid thatselectively hybridizes to AAV5 can be of longer length than the AAV5genome, it can be about the same length as the AAV5 genome or it can beshorter than the AAV5 genome. The length of the nucleic acid is limitedon the shorter end of the size range only by its specificity forhybridization to AAV5, i.e., once it is too short, typically less thanabout 5 to 7 nucleotides in length, it will no longer bind specificallyto AAV5, but rather will hybridize to numerous background nucleic acids.Additionally contemplated by this invention is a nucleic acid that has aportion that specifically hybridizes to AAV5 and a portion thatspecifically hybridizes to a gene of interest inserted within AAV5.

The present invention further provides an isolated nucleic acid encodingan adeno-associated virus 5 Rep protein. The AAV5 Rep proteins areencoded by open reading frame (ORF) 1 of the AAV5 genome. Examples ofthe AAV5 Rep genes are shown in the nucleic acid set forth in SEQ IDNO:1, and include nucleic acids consisting essentially of the nucleotidesequences set forth in SEQ ID NOS: 10 (Rep52), 11 (Rep78), 13 (Rep40),and 15 (Rep68), and nucleic acids comprising the nucleotide sequencesset forth in SEQ ID NOS: 10, 11, 13, and 15. However, the presentinvention contemplates that the Rep nucleic acid can include any one,two, three, or four of the four Rep proteins, in any order, in such anucleic acid. Furthermore, minor modifications are contemplated in thenucleic acid, such as silent mutations in the coding sequences,mutations that make neutral or conservative changes in the encoded aminoacid sequence, and mutations in regulatory regions that do not disruptthe expression of the gene. Examples of other minor modifications areknown in the art. Further modifications can be made in the nucleic acid,such as to disrupt or alter expression of one or more of the Repproteins in order to, for example, determine the effect of such adisruption; such as to mutate one or more of the Rep proteins todetermine the resulting effect, etc. However, in general, a modifiednucleic acid encoding a Rep protein will have at least about 85%, about90%, about 93%, about 95%, about 98% or 100% homology to the Rep nucleicsequences described herein e.g., SEQ ID NOS: 10, 11, 13 and 15, and theRep polypeptide encoded therein will have overall about 93%, about 95%,about 98%, about 99% or 100% homology with the amino acid sequencedescribed herein, e.g., SEQ ID NOS:2, 3, 12 and 14. Percent homology isdetermined by the techniques described herein.

The present invention also provides an isolated nucleic acid thatselectively or specifically hybridizes with a nucleic acid consistingessentially of the nucleotide sequence set forth in SEQ ID NOS:10, 11,13 and 15, and an isolated nucleic acid that selectively hybridizes witha nucleic acid comprising the nucleotide sequence set forth in SEQ IDNOS:10, 11, 13 and 15. “Selectively hybridizing” and “stringency ofhybridization” is defined elsewhere herein.

As described above, the present invention provides the nucleic acidencoding a Rep 40 protein and, in particular an isolated nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO: 13, anisolated nucleic acid consisting essentially of the nucleotide sequenceset forth in SEQ ID NO: 13, and a nucleic acid encoding theadeno-associated virus 5 protein having the amino acid sequence setforth in SEQ ID NO: 12. The present invention also provides the nucleicacid encoding a Rep 52 protein, and in particular an isolated nucleicacid comprising the nucleotide sequence set forth in SEQ ID NO:10, anisolated nucleic acid consisting essentially of the nucleotide sequenceset forth in SEQ ID NO:10, and a nucleic acid encoding theadeno-associated virus 5 Rep protein having the amino acid sequence setforth in SEQ ID NO:2. The present invention further provides the nucleicacid encoding a Rep 68 protein and, in particular an isolated nucleicacid comprising the nucleotide sequence set forth in SEQ ID NO: 15, anisolated nucleic acid consisting essentially of the nucleotide sequenceset forth in SEQ ID NO: 15, and a nucleic acid encoding theadeno-associated virus 5 protein having the amino acid sequence setforth in SEQ ID NO: 14. And, further, the present invention provides thenucleic acid encoding a Rep 78 protein, and in particular an isolatednucleic acid comprising the nucleotide sequence set forth in SEQ IDNO:11, an isolated nucleic acid consisting essentially of the nucleotidesequence set forth in SEQ ID NO:11, and a nucleic acid encoding theadeno-associated virus 5 Rep protein having the amino acid sequence setforth in SEQ ID NO:3. As described elsewhere herein, these nucleic acidscan have minor modifications, including silent nucleotide substitutions,mutations causing conservative amino acid substitutions in the encodedproteins, and mutations in control regions that do not or minimallyaffect the encoded amino acid sequence.

The present invention further provides a nucleic acid encoding theentire AAV5 Capsid polypeptide. Furthermore, the present inventionprovides a nucleic acid encoding each of the three AAV5 coat proteins,VP 1, VP2, and VP3. Thus, the present invention provides a nucleic acidencoding AAV5 VP 1, a nucleic acid encoding AAV5 VP2, and a nucleic acidencoding AAV5 VP3. Thus, the present invention provides a nucleic acidencoding the amino acid sequence set forth in SEQ ID NO:4 (VP 1); anucleic acid encoding the amino acid sequence set forth in SEQ ID NO:5(VP2), and a nucleic acid encoding the amino acid sequence set forth inSEQ ID NO:6 (VP3). The present invention also specifically provides anucleic acid comprising SEQ ID NO:7 (VP1 gene); a nucleic acidcomprising SEQ ID NO:8 (VP2 gene); and a nucleic acid comprising SEQ IDNO:9 (VP3 gene). The present invention also specifically provides anucleic acid consisting essentially of SEQ ID NO:7 (VP1 gene), a nucleicacid consisting essentially of SEQ ID NO:8 (VP2 gene), and a nucleicacid consisting essentially of SEQ ID NO:9 (VP3 gene). Minormodifications in the nucleotide sequences encoding the capsid, or coat,proteins are contemplated, as described above for other AAV5 nucleicacids. However, in general, a modified nucleic acid encoding a capsidprotein will have at least about 85%, about 90%, about 93%, about 95%,about 98% or 100% homology to the capsid nucleic sequences describedherein e.g., SEQ ID NOS: 7, 8, and 9, and the capsid polypeptide encodedtherein will have overall about 93%, about 95%, about 98%, about 99% or100% homology with the amino acid sequence described herein, e.g., SEQID NOS:4, 5, and 6. Nucleic acids that selectively hybridize with thenucleic acids of SEQ ID NOS:7,8 and 9 under the conditions describedabove are also provided.

The present invention also provides a cell containing one or more of theherein described nucleic acids, such as the AAV5 genome, AAV5 ORF1 andORF2, each AAV5 Rep protein gene, or each AAV5 capsid protein gene. Sucha cell can be any desired cell and can be selected based upon the useintended. For example, cells can include bacterial cells, yeast cells,insect cells, human HeLa cells and simian Cos cells as well as otherhuman and mammalian cells and cell lines. Primary cultures as well asestablished cultures and cell lines can be used. Nucleic acids of thepresent invention can be delivered into cells by any selected means, inparticular depending upon the target cells. Many delivery means arewell-known in the art. For example, electroporation, calcium phosphateprecipitation, microinjection, cationic or anionic liposomes, andliposomes in combination with a nuclear localization signal peptide fordelivery to the nucleus can be utilized, as is known in the art.Additionally, if the nucleic acids are in a viral particle, the cellscan simply be transduced with the virion by standard means known in theart for AAV transduction. Small amounts of the recombinant AAV5 viruscan be made to infect cells and produce more of itself.

The invention provides purified AAV5 polypeptides. The term“polypeptide” as used herein refers to a polymer of amino acids andincludes full-length proteins and fragments thereof. Thus, “protein,polypeptide,” and “peptide” are often used interchangeably herein.Substitutions can be selected by known parameters to be neutral (see,e.g., Robinson W E Jr, and Mitchell W M., AIDS 4:S151-S162 (1990)). Aswill be appreciated by those skilled in the art, the invention alsoincludes those polypeptides having slight variations in amino acidsequences or other properties. Such variations may arise naturally asallelic variations (e.g., due to genetic polymorphism) or may beproduced by human intervention (e.g., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutants. Minor changes in amino acid sequence are generally preferred,such as conservative amino acid replacements, small internal deletionsor insertions, and additions or deletions at the ends of the molecules.Substitutions may be designed based on, for example, the model ofDayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat'lBiomed. Res. Found., Washington, D.C.). These modifications can resultin changes in the amino acid sequence, provide silent mutations, modifya restriction site, or provide other specific mutations. The location ofany modifications to the polypeptide will often determine its impact onfunction. Particularly, alterations in regions non-essential to proteinfunction will be tolerated with fewer effects on function. Elsewhere inthe application regions of the AAV5 proteins are described to provideguidance as to where substitutions, additions or deletions can be madeto minimize the likelihood of disturbing the function of the variant.

A polypeptide of the present invention can be readily obtained by any ofseveral means. For example, the polypeptide of interest can besynthesized chemically by standard methods. Additionally, the codingregions of the genes can be recombinantly expressed and the resultingpolypeptide isolated by standard methods. Furthermore, an antibodyspecific for the resulting polypeptide can be raised by standard methods(see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988), and theprotein can be isolated from a cell expressing the nucleic acid encodingthe polypeptide by selective hybridization with the antibody. Thisprotein can be purified to the extent desired by standard methods ofprotein purification (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

Typically, to be unique, a polypeptide fragment of the present inventionwill be at least about 5 amino acids in length; however, uniquefragments can be 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ormore amino acids in length. A unique polypeptide will typically comprisesuch a unique fragment; however, a unique polypeptide can also bedetermined by its overall homology. A unique polypeptide can be 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids inlength. Uniqueness of a polypeptide fragment can readily be determinedby standard methods such as searches of computer databases of knownpeptide or nucleic acid sequences or by hybridization studies to thenucleic acid encoding the protein or to the protein itself, as known inthe art. The uniqueness of a polypeptide fragment can also be determinedimmunologically as well as functionally. Uniqueness can be simplydetermined in an amino acid-by-amino acid comparison of thepolypeptides.

An antigenic or immunoreactive fragment of this invention is typicallyan amino acid sequence of at least about 5 consecutive amino acids, andit can be derived from the AAV5 polypeptide amino acid sequence. Anantigenic AAV5 fragment is any fragment unique to the AAV5 protein, asdescribed herein, against which an AAV5-specific antibody can be raised,by standard methods. Thus, the resulting antibody-antigen reactionshould be specific for AAV5.

The present invention provides an isolated AAV5 Rep protein. An AAV5 Reppolypeptide is encoded by ORF1 of AAV5. The present invention alsoprovides each individual AAV5 Rep protein. Thus the present inventionprovides AAV5 Rep 40 (e.g., SEQ ID NO: 12), or a unique fragmentthereof. The present invention provides AAV5 Rep 52 (e.g., SEQ ID NO:2), or a unique fragment thereof. The present invention provides AAV5Rep 68 (e.g., SEQ ID NO: 14), or a unique fragment thereof.

The present invention provides an example of AAV5 Rep 78 (e.g., SEQ IDNO: 3), or a unique fragment thereof. By “unique fragment thereof” ismeant any smaller polypeptide fragment encoded by an AAV5 rep gene thatis of sufficient length to be found only in the Rep polypeptide.Substitutions and modifications of the amino acid sequence can be madeas described above and, further, can include protein processingmodifications, such as glycosylation, to the polypeptide.

The present invention further provides an AAV5 Capsid polypeptide or aunique fragment thereof. AAV5 capsid polypeptide is encoded by ORF 2 ofAAV5.

The present invention further provides the individual AAV5 capsidproteins, VP 1, VP2 and VP3 or unique fragments thereof. Thus, thepresent invention provides an isolated polypeptide having the amino acidsequence set forth in SEQ ID NO:4 (VP 1). The present inventionadditionally provides an isolated polypeptide having the amino acidsequence set forth in SEQ ID NO:5 (VP2). The present invention alsoprovides an isolated polypeptide having the amino acid sequence setforth in SEQ ID NO:6 (VP3). By “unique fragment thereof” is meant anysmaller polypeptide fragment encoded by any AAV5 capsid gene that is ofsufficient length to be found only in the AAV5 capsid protein.Substitutions and modifications of the amino acid sequence can be madeas described above and, further, can include protein processingmodifications, such as glycosylation, to the polypeptide. However, anAAV5 Capsid polypeptide including all three coat proteins will havegreater than about 56% overall homology to the polypeptide encoded bythe nucleotides set forth in SEQ ID NOS:4, 5 or 6. The protein can haveabout 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 93%,95%, 97% or even 100% homology to the amino acid sequence encoded by thenucleotides set forth in SEQ ID NOS:4, 5 or 6. An AAV5 VP1 polypeptidecan have at least about 58%, about 60%, about 70%, about 80%, about 90%,93%, 95%, 97% or about 100% homology to the amino acid sequence setforth in SEQ ID NO:4. An AAV5 VP2 polypeptide can have at least about58%, about 60%, about 70%, about 80%, about 90%, 93%, 95%, 97% or about100% homology to the amino acid sequence set forth in SEQ ID NO:5. AnAAV5 VP3 polypeptide can have at least about 60%, about 70%, about 80%,about 90%, 93%, 95%, 97% or about 100% homology to the amino acidsequence set forth in SEQ ID NO:6.

The present invention further provides an isolated antibody thatspecifically binds an AAV5 Rep protein or a unique epitope thereof. Alsoprovided are isolated antibodies that specifically bind the AAV5 Rep 52protein, the AAV5 Rep 40 protein, the AAV5 Rep 68 protein and the AAV5Rep 78 protein having the amino acid sequences set forth in SEQ ID NO:2,SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 3, respectively or thatspecifically binds a unique fragment thereof. Clearly, any givenantibody can recognize and bind one of a number of possible epitopespresent in the polypeptide; thus only a unique portion of a polypeptide(having the epitope) may need to be present in an assay to determine ifthe antibody specifically binds the polypeptide.

The present invention additionally provides an isolated antibody thatspecifically binds any of the adeno-associated virus 5 Capsid proteins(VP1, VP2 or VP3), a unique epitope thereof, or the polypeptidecomprising all three AAV5 coat proteins. Also provided is an isolatedantibody that specifically binds the AAV5 capsid protein having theamino acid sequence set forth in SEQ ID NO:4 (VP 1), or thatspecifically binds a unique fragment thereof. The present inventionfurther provides an isolated antibody that specifically binds the AAV5Capsid protein having the amino acid sequence set forth in SEQ ID NO:5(VP2), or that specifically binds a unique fragment thereof. Theinvention additionally provides an isolated antibody that specificallybinds the AAV5 Capsid protein having the amino acid sequence set forthin SEQ ID NO:6 (VP3), or that specifically binds a unique fragmentthereof. Again, any given antibody can recognize and bind one of anumber of possible epitopes present in the polypeptide; thus only aunique portion of a polypeptide (having the epitope) may need to bepresent in an assay to determine if the antibody specifically binds thepolypeptide.

The antibody can be a component of a composition that comprises anantibody that specifically binds the AAV5 protein. The composition canfurther comprise, e.g., serum, serum-free medium, or a pharmaceuticallyacceptable carrier such as physiological saline, etc.

By “an antibody that specifically binds” an AAV5 polypeptide or proteinis meant an antibody that selectively binds to an epitope on any portionof the AAV5 peptide such that the antibody binds specifically to thecorresponding AAV5 polypeptide without significant background. Specificbinding by an antibody further means that the antibody can be used toselectively remove the target polypeptide from a sample comprising thepolypeptide or and can readily be determined by radioimmunoassay (RIA),bioassay, or enzyme-linked immunosorbant (ELISA) technology. An ELISAmethod effective for the detection of the specific antibody-antigenbinding can, for example, be as follows: (1) bind the antibody to asubstrate; (2) contact the bound antibody with a sample containing theantigen; (3) contact the above with a secondary antibody bound to adetectable moiety (e.g., horseradish peroxidase enzyme or alkalinephosphatase enzyme); (4) contact the above with the substrate for theenzyme; (5) contact the above with a color reagent; (6) observe thecolor change.

An antibody can include antibody fragments such as Fab fragments whichretain the binding activity. Antibodies can be made as described in,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1988). Briefly, purifiedantigen can be injected into an animal in an amount and in intervalssufficient to elicit an immune response. Antibodies can either bepurified directly, or spleen cells can be obtained from the animal. Thecells are then fused with an immortal cell line and screened forantibody secretion. Individual hybridomas are then propagated asindividual clones serving as a source for a particular monoclonalantibody.

The present invention additionally provides a method of screening a cellfor infectivity by AAV5 comprising contacting the cell with AAV5 anddetecting the presence of AAV5 in the cells. AAV5 particles can bedetected using any standard physical or biochemical methods. Forexample, physical methods that can be used for this detection includeDNA based methods such as 1) polymerase chain reaction (PCR) for viralDNA or RNA or 2) direct hybridization with labeled probes, andimmunological methods such as by 3) antibody directed against the viralstructural or non-structural proteins. Catalytic methods of viraldetection include, but are not limited to, detection of site and strandspecific DNA nicking activity of Rep proteins or replication of an AAVorigin-containing substrate. Reporter genes can also be utilized todetect cells that transduct AAV-5. For example, β-gal, green flourescentprotein or luciferase can be inserted into a recombinant AAV-5. The cellcan then be contacted with the recombinant AAV-5, either in vitro or invivo and a colorimetric assay could detect a color change in the cellsthat would indicate transduction of AAV-5 in the cell. Additionaldetection methods are outlined in Fields, Virology, Raven Press, NewYork, N.Y. 1996.

For screening a cell for infectivity by AAV5, wherein the presence ofAAV5 in the cells is determined by nucleic acid hybridization methods, anucleic acid probe for such detection can comprise, for example, aunique fragment of any of the AAV5 nucleic acids provided herein. Theuniqueness of any nucleic acid probe can readily be determined asdescribed herein. Additionally, the presence of AAV5 in cells can bedetermined by flourescence, antibodies to gene products, focus formingassays, plaque lifts, Western blots and chromogenic assays. The nucleicacid can be, for example, the nucleic acid whose nucleotide sequence isset forth in SEQ ID NO: 1, 7, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20,21, 22, 23 or a unique fragment thereof.

The present invention includes a method of determining the suitabilityof an AAV5 vector for administration to a subject comprisingadministering to an antibody-containing sample from the subject anantigenic fragment of an isolated AAV5 Rep or Capsid protein, anddetecting neutralizing antibody-antigen reaction in the sample, thepresence of a neutralizing reaction indicating the AAV5 vector may beunsuitable for use in the subject. The present method of determining thesuitability of an AAV5 vector for administration to a subject cancomprise contacting an antibody-containing sample from the subject witha unique antigenic or immunogenic fragment of an AAV5 Rep protein (e.g.Rep 40, Rep 52, Rep 68, Rep 78) and detecting an antibody-antigenreaction in the sample, the presence of a reaction indicating the AAV5vector to be unsuitable for use in the subject. The AAV5 Rep proteinsare provided herein, and their antigenic fragments are routinelydetermined. The AAV5 capsid protein can be used to select an antigenicor immunogenic fragment, for example from the amino acid sequence setforth in SEQ ID NO:4 (VP1), the amino acid sequence set forth in SEQ IDNO: 5 (VP2) or the amino acid sequence set forth in SEQ ID NO:6 (VP3).Alternatively, or additionally, an antigenic or immunogenic fragment ofan isolated AAV5 Rep protein can be utilized in this determinationmethod. The AAV5 Rep protein from which an antigenic fragment isselected can have the amino acid sequence encoded by the nucleic acidset forth in SEQ ID NO:1, the amino acid sequence set forth in SEQ IDNO:2, or the amino acid sequence set forth in SEQ ID NO:3, the aminoacid sequence set forth in SEQ ID NO: 12, or the amino acid sequence setforth in SEQ ID NO:14.

The AAV5 polypeptide fragments can be analyzed to determine theirantigenicity, immunogenicity and/or specificity. Briefly, variousconcentrations of a putative immunogenically specific fragment areprepared and administered to a subject and the immunological response(e.g., the production of antibodies or cell mediated immunity) of ananimal to each concentration is determined. The amounts of antigenadministered depend on the subject, e.g. a human, rabbit or a guineapig, the condition of the subject, the size of the subject, etc.Thereafter an animal so inoculated with the antigen can be exposed tothe AAV5 viral particle or AAV5 protein to test the immunoreactivity orthe antigenicity of the specific immunogenic fragment. The specificityof a putative antigenic or immunogenic fragment can be ascertained bytesting sera, other fluids or lymphocytes from the inoculated animal forcross reactivity with other closely related viruses, such as AAV1, AAV2,AAV3, AAV4 and AAV5.

The hemagglutination assay can also be used to rapidly identify anddetect AAV5 viral particles. Detection of hemagglutination activitycorrelates with infectivity and can be used to titer the virus. Thisassay could also be used to identify antibodies in a patients serumwhich might interact with the virus. Hemagglutination has been shown tocorrelate with infectivity and therefore hemagglutination may be auseful assay for identify cellular receptors for AAV5.

By the “suitability of an AAV5 vector for administration to a subject”is meant a determination of whether the AAV5 vector will elicit aneutralizing immune response upon administration to a particularsubject. A vector that does not elicit a significant immune response isa potentially suitable vector, whereas a vector that elicits asignificant, neutralizing immune response (e.g. at least 90%) is thuslikely to be unsuitable for use in that subject. Significance of anydetectable immune response is a standard parameter understood by theskilled artisan in the field. For example, one can incubate thesubject's serum with the virus, then determine whether that virusretains its ability to transduce cells in culture. If such virus cannottransduce cells in culture, the vector likely has elicited a significantimmune response.

Alternatively, or additionally, one skilled in the art could determinewhether or not AAV5 administration would be suitable for a particularcell type of a subject. For example, the artisan could culture musclecells in vitro and transduce the cells with AAV5 in the presence orabsence of the subject's serum. If there is a reduction in transductionefficiency, this could indicate the presence of a neutralizing antibodyor other factors that may inhibit transduction. Normally, greater than90% inhibition would have to be observed in order to rule out the use ofAAV-5 as a vector. However, this limitation could be overcome bytreating the subject with an immunosuppressant that could block thefactors inhibiting transduction.

As will be recognized by those skilled in the art, numerous types ofimmunoassays are available for use in the present invention to detectbinding between an antibody and an AAV5 polypeptide of this invention.For instance, direct and indirect binding assays, competitive assays,sandwich assays, and the like, as are generally described in, e.g., U.S.Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157;3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, N.Y. (1988). Forexample, enzyme immunoassays such as immunofluorescence assays (IFA),enzyme linked immunosorbent assays (ELISA) and immunoblotting can bereadily adapted to accomplish the detection of the antibody. An ELISAmethod effective for the detection of the antibody bound to the antigencan, for example, be as follows: (1) bind the antigen to a substrate;(2) contact the bound antigen with a fluid or tissue sample containingthe antibody; (3) contact the above with a secondary antibody specificfor the antigen and bound to a detectable moiety (e.g., horseradishperoxidase enzyme or alkaline phosphatase enzyme); (4) contact the abovewith the substrate for the enzyme; (5) contact the above with a colorreagent; (6) observe color change.

The antibody-containing sample of this method can comprise anybiological sample which would contain the antibody or a cell containingthe antibody, such as blood, plasma, serum, bone marrow, saliva andurine.

The present invention also provides a method of producing the AAV5 virusby transducing a cell with the nucleic acid encoding the virus.

The present method further provides a method of delivering an exogenous(heterologous) nucleic acid to a cell comprising administering to thecell an AAV5 particle containing a vector comprising the nucleic acidinserted between a pair of AAV inverted terminal repeats, therebydelivering the nucleic acid to the cell.

The AAV ITRs in the vector for the herein described delivery methods canbe AAV5 ITRs (SEQ ID NOS: 19 and 20). Furthermore, the AAV ITRs in thevector for the herein described nucleic acid delivery methods can alsocomprise AAV1, AAV2, AAV3, AAV4, or AAV6 inverted terminal repeats.

The present invention also includes a method of delivering aheterologous nucleic acid to a subject comprising administering to acell from the subject an AAV5 particle containing a vector comprisingthe nucleic acid inserted between a pair of AAV inverted terminalrepeats, and returning the cell to the subject, thereby delivering thenucleic acid to the subject. The AAV ITRs can be any AAV ITRs, includingAAV5 ITRs and AAV2 ITRs. For example, in an ex vivo administration,cells are isolated from a subject by standard means according to thecell type and placed in appropriate culture medium, again according tocell type (see, e.g., ATCC catalog). Viral particles are then contactedwith the cells as described above, and the virus is allowed to transducethe cells. Cells can then be transplanted back into the subject's body,again by means standard for the cell type and tissue (e.g., in general,U.S. Pat. No. 5,399,346; for neural cells, Dunnett, S. B. and Björklund,A., eds., Transplantation: Neural Transplantation-A Practical Approach,Oxford University Press, Oxford (1992)). If desired, prior totransplantation, the cells can be studied for degree of transduction bythe virus, by known detection means and as described herein. Cells forex vivo transduction followed by transplantation into a subject can beselected from those listed above, or can be any other selected cell.Preferably, a selected cell type is examined for its capability to betransfected by AAV5. Preferably, the selected cell will be a cellreadily transduced with AAV5 particles; however, depending upon theapplication, even cells with relatively low transduction efficienciescan be useful, particularly if the cell is from a tissue or organ inwhich even production of a small amount of the protein or antisense RNAencoded by the vector will be beneficial to the subject.

The present invention further provides a method of delivering a nucleicacid to a cell in a subject comprising administering to the subject anAAV5 particle containing a vector comprising the nucleic acid insertedbetween a pair of AAV inverted terminal repeats, thereby delivering thenucleic acid to a cell in the subject. Administration can be an ex vivoadministration directly to a cell removed from a subject, such as any ofthe cells listed above, followed by replacement of the cell back intothe subject, or administration can be in vivo administration to a cellin the subject. For ex vivo administration, cells are isolated from asubject by standard means according to the cell type and placed inappropriate culture medium, again according to cell type (see, e.g.,ATCC catalog). Viral particles are then contacted with the cells asdescribed above, and the virus is allowed to transfect the cells. Cellscan then be transplanted back into the subject's body, again by meansstandard for the cell type and tissue (e.g., for neural cells, Dunnett,S. B. and Björklund, A., eds., Transplantation. Neural Transplantation-APractical Approach, Oxford University Press, Oxford (1992)). If desired,prior to transplantation, the cells can be studied for degree oftransfection by the virus, by known detection means and as describedherein.

The present invention further provides a method of delivering a nucleicacid to a cell in a subject having neutralizing antibodies to AAV2comprising administering to the subject an AAV5 particle containing avector comprising the nucleic acid, thereby delivering the nucleic acidto a cell in the subject. A subject that has neutralizing antibodies toAAV2 can readily be determined by any of several known means, such ascontacting AAV2 protein(s) with an antibody-containing sample, such asblood, from a subject and detecting an antigen-antibody reaction in thesample. Delivery of the AAV5 particle can be by either ex vivo or invivo administration as herein described. Thus, a subject who might havean adverse immunogenic reaction to a vector administered in an AAV2viral particle can have a desired nucleic acid delivered using an AAV5particle. This delivery system can be particularly useful for subjectswho have received therapy utilizing AAV2 particles in the past and havedeveloped antibodies to AAV2. An AAV5 regimen can now be substituted todeliver the desired nucleic acid.

In any of the methods of delivering heterologous nucleic acids to a cellor subject described herein, the AAV5-conjugated nucleic acid or AAV5particle-conjugated nucleic acids described herein can be used.

In vivo administration to a human subject or an animal model can be byany of many standard means for administering viruses, depending upon thetarget organ, tissue or cell. Virus particles can be administeredorally, parenterally (e.g., intravenously), by intramuscular injection,by direct tissue or organ injection, by intraperitoneal injection,topically, transdermally, via aerosol delivery, via the mucosa or thelike. Viral nucleic acids (non-encapsidated) can also be administered,e.g., as a complex with cationic liposomes, or encapsulated in anionicliposomes. The present compositions can include various amounts of theselected viral particle or non-encapsidated viral nucleic acid incombination with a pharmaceutically acceptable carrier and, in addition,if desired, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, diluents, etc. Parental administration, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Dosages will depend upon the mode of administration,the disease or condition to be treated, and the individual subject'scondition, but will be that dosage typical for and used inadministration of other AAV vectors, such as AAV2 vectors. Often asingle dose can be sufficient; however, the dose can be repeated ifdesirable.

Administration methods can be used to treat brain disorders such asParkinson's disease, Alzheimer's disease, and demyelination disease.Other diseases that can be treated by these methods include metabolicdisorders such as, muscoloskeletal diseases, cardiovascular disease,cancer, and autoimmune disorders.

Administration of this recombinant AAV5 virion to the cell can beaccomplished by any means, including simply contacting the particle,optionally contained in a desired liquid such as tissue culture medium,or a buffered saline solution, with the cells. The virion can be allowedto remain in contact with the cells for any desired length of time, andtypically the virion is administered and allowed to remain indefinitely.For such in vitro methods, the virion can be administered to the cell bystandard viral transduction methods, as known in the art and asexemplified herein. Titers of virus to administer can vary, particularlydepending upon the cell type, but will be typical of that used for AAVtransduction in general which is well known in the art. Additionally thetiters used to transduce the particular cells in the present examplescan be utilized.

The cells that can be transduced by the present recombinant AAV5 virioncan include any desired cell, such as the following cells and cellsderived from the following tissues, human as well as other mammaliantissues, such as primate, horse, sheep, goat, pig, dog, rat, and mouse:Adipocytes, Adenocyte, Adrenal cortex, Amnion, Aorta, Ascites,Astrocyte, Bladder, Bone, Bone marrow, Brain, Breast, Bronchus, Cardiacmuscle, Cecum, Cervix, Chorion, Colon, Conjunctiva, Connective tissue,Cornea, Dermis, Duodenum, Endometrium, Endothelium, Endothelial cells,Epithelial tissue, Epithelial cells, Epidermis, Esophagus, Eye, Fascia,Fibroblasts, Foreskin, Gastric, Glial cells, Glioblast, Gonad, Hepaticcells, Histocyte, Ileum, Intestine, small Intestine, Jejunum,Keratinocytes, Kidney, Larynx, Leukocytes, Lipocyte, Liver, Lung, Lymphnode, Lymphoblast, Lymphocytes, Macrophages, Mammary alveolar nodule,Mammary gland, Mastocyte, Maxilla, Melanocytes, Mesenchymal, Monocytes,Mouth, Myelin, Myoblasts Nervous tissue, Neuroblast, Neurons, Neuroglia,Osteoblasts, Osteogenic cells, Ovary, Palate, Pancreas, Papilloma,Peritoneum, Pituicytes, Pharynx, Placenta, Plasma cells, Pleura,Prostate, Rectum, Salivary gland, Skeletal muscle, Skin, Smooth muscle,Somatic, Spleen, Squamous, Stomach, Submandibular gland, Submaxillarygland, Synoviocytes, Testis, Thymus, Thyroid, Trabeculae, Trachea,Turbinate, Umbilical cord, Ureter, and Uterus.

Statement of Utility

The present invention provides recombinant vectors based on AAV5. Suchvectors may be useful for transducing erythroid progenitor cells orcells lacking heparin sulfate proteoglycans which is very inefficientwith AAV2 based vectors. These vectors may also be useful fortransducing cells with a nucleic acid of interest in order to producecell lines that could be used to screen for agents that interact withthe gene product of the nucleic acid of interest. In addition totransduction of other cell types, transduction of erythroid cells wouldbe useful for the treatment of cancer and genetic diseases which can becorrected by bone marrow transplants using matched donors. Some examplesof this type of treatment include, but are not limited to, theintroduction of a therapeutic gene such as genes encoding interferons,interleukins, tumor necrosis factors, adenosine deaminase, cellulargrowth factors such as lymphokines, blood coagulation factors such asfactor VIII and IX, cholesterol metabolism uptake and transport proteinsuch as EpoE and LDL receptor, and antisense sequences to inhibit viralreplication of, for example, hepatitis or HIV.

The present invention provides a vector comprising the AAV5 virus aswell as AAV5 viral particles. While AAV5 is similar to AAV2, the twoviruses are found herein to be physically and genetically distinct.These differences endow AAV5 with some unique advantages which bettersuit it as a vector for gene therapy.

Furthermore, as shown herein, AAV5 capsid protein is distinct from AAV2capsid protein and exhibits different tissue tropism. AAV2 and AAV5likely utilize distinct cellular receptors. AAV2 and AAV5 areserologically distinct and thus, in a gene therapy application, AAV5would allow for transduction of a patient who already possessneutralizing antibodies to AAV2 either as a result of naturalimmunological defense or from prior exposure to AAV2 vectors.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

To understand the nature of AAV5 virus and to determine its usefulnessas a vector for gene transfer, it was cloned and sequenced.

Cell Culture and Virus Propagation

Cos and HeLa cells were maintained as monolayer cultures in D10 medium(Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100μg/ml penicillin, 100 units/ml streptomycin and IX Fungizone asrecommended by the manufacturer; (GIBCO, Gaithersburg, Md., USA). Allother cell types were grown under standard conditions which have beenpreviously reported.

Virus was produced as previously described for AAV2 using the Betagalactosidase vector plasmid and a helper plasmid containing the AAV5Rep and Cap genes (9). The helper plasmid was constructed in such a wayto minimize any homologous sequence between the helper and vectorplasmids. This step was taken to minimize the potential for wield-type(wt) particle formation by homologous recombination.

DNA Cloning and Sequencing and Analysis

In order to clone the genome of AAV5, infectious cell lysate wasexpanded in adherent cos cells and then suspension HeLa cells with theresulting viral particles isolated by CsCl isopynic gradientcentrifugation. DNA dot blots of Aliquots of the gradient fractionsindicated that the highest concentration of viral genomes were containedin fractions with a refractive index of approx. 1.372. While the initialdescription of the virus did not determine the density of the particles,this value is similar to that of AAV2. Analysis of annealed virionderived DNA obtained from these fractions indicated a major species of4.6 kb in length which upon restriction analysis gave bands similar insize to those previously reported. Additional restriction mappingindicated a unique BssHII site at one end of the viral genome. This sitewas used to clone the major fragment of the viral genome. Additionaloverlapping clones were isolated and the sequence determined. Twodistinct open reading frames (ORF) were identified. Computer analysisindicated that the left-hand ORF is approx 60% similar to that of theRep gene of AAV2. Of the 4 other reported AAV serotypes, all havegreater than 90% similarity in this ORF. The right ORF of the viralcapsid proteins is also approximately 60% homologous to the Capsid ORFof AAV2. As with other AAV serotypes reported, the divergence betweenAAV5 and AAV2 is clustered in multiple blocks. By using the publishedthree dimensional structure of the canine parvovirus and computer aidedsequence comparisons, a number of these divergent regions have beenshown to be on the exterior of the virus and thus suggest an alteredtissue tropism.

Within the p5 promoter, a number of the core transcriptional elementsare conserved such as the tataa box and YY1 site around thetranscriptional start site. However the YY1 site at −60 and the upstreamE-Box elements are not detectable suggesting an alternative method ofregulation or activation.

The inverted terminal repeats (ITRs) of the virus were cloned as afragment from the right end of the genome. The resulting fragment wasfound to contain a number of sequence changes compared to AAV2. However,these changes were found to be complementary and did not affect theability of this region to fold into a hairpin structure. Within the stemregion of the hairpin two sequence elements have been found to becritical for the function of the ITRs as origins of viral replication. Arepeat motif of GAGC/T which serves as the recognition site of Rep and aGGTTGAG sequence downstream of the Rep binding site which is theposition of Rep's site and strand specific cleavage reaction. Thissequence is not conserved between AAV5 and the other cloned AAV'ssuggesting that the ITRs and Rep proteins of AAV5 cannot compliment theother known AAV's.

To test the cross complementarity of AAV2 ITR containing genome and AAV5ITR containing genomes recombinant particles were packaged either usingtype 2 Rep and Cap or type 5 Rep and Cap expression plasmids aspreviously described. As shown in FIG. 2, viral particles were producedonly when the respective expression plasmids were used to package thecognate ITRs. This result is distinct from that of other serotypes ofAAV which have shown cross complementary in packaging.

This specificity of AAV5 Rep for AAV5 ITRs was confirmed using aterminal resolution assay which can identify the site within one ITRcleaved by the Rep protein. Incubation of the Type 5 Rep protein with atype 2 ITR did not produce any cleavage products. In contrast, additionof type 2 Rep cleaved the DNA at the expected site. However AAV5 Rep didproduce cleavage products when incubated with a type 5 ITR. The sitemapped to a region 21 bases from the Rep binding motif that is similarto AAV2 TRS. The site in AAV2 is CGGT TGAG (SEQ ID NO: 22) but in type 5ITR is CGGT GTGA (SEQ ID NO: 21). The ability of AAV5 Rep to cleave at adifferent but similarly positioned site may result in integration ofAAV5 at a distinct chromosomal locus compared to AAV2.

Recombinant virus produced using AAV5 Rep and Cap was obtained at agreater titer than type 2. For example, in a comparative study, viruswas isolated from 8×10⁷ COS cells by CsCl banding and the distributionof the Beta galactosidase genomes across the gradient were determined byDNA dot blots of aliquots of gradient fractions. DNA dot blot titersindicated that AAV5 particles were produced at a 10–50 fold higher levelthan AAV2.

The sequence divergence in the capsid protein ORF implies that thetissue tropism of AAV2 and AAV5 would differ. To study the transductionefficiency of AAV5 and AAV2, a variety of cell lines were transducedwith serial dilution's of the purified virus expressing the gene fornuclear localized Beta galactosidase activity. Approx. 2×10⁴ cells wereexposed to virus in 1 ml of serum containing media for a period of 48–60hrs. After this time the cells were fixed and stained forBeta-galactosidase activity with5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside (Xgal) (ICNBiochemicals). Biological titers were determined by counting the numberof positive cells in the different dilutions using a calibratedmicroscope ocular then multiplying by the area of the well. Titers weredetermined by the average number of cells in a minimum of 10fields/well. Transduction of cos, HeLa, and 293, and IB3 cells with asimilar number of particles showed approximately 10 fold decrease intiter with AAV5 compared with AAV2. In contrast MCF7 cells showed a50–100 fold difference in transduction efficiency. Furthermore, bothvectors transduced NIH 3T3 cells relatively poorly.

A recent publication reported that heparin proteoglycans on the surfaceof cells are involved in viral transduction. Addition of soluble heparinhas been shown to inhibit transduction by blocking viral binding. Sincethe transduction data suggested a difference in tissue tropism for AAV5and AAV2, the sensitivity of AAV5 transduction to heparin wasdetermined. At an MOI of 100, the addition of 20 μg/ml of heparin had noeffect on AAV5 transduction. In contrast this amount of heparininhibited 90% of the AAV2 transduction. Even at an MOI of 1000, noinhibition of AAV5 transduction was detected. These data support theconclusions of the tissue tropism study, i.e. that AAV2 and AAV5 mayutilize a distinct cell surface molecules and therefore the mechanism ofuptake may differ as well.

AAV5 is a distinct virus within the dependovirus family based onsequence analysis, tissue tropism, and sensitivity to heparin. Whileelements of the P5 promoter are retained between AAV2–6 some elementsare absent in AAV5 suggesting alternative mechanism of regulation. TheITR and Rep ORF are distinct from those previously identified and failto complement the packaging of AAV2 based genomes. The ITR of AAV5contains a different TRS compared to other serotypes of AAV which isresponsible for the lack of complementation of the ITRs. This unique TRSshould also result in a different integration locus for AAV5 compared tothat of AAV2. Furthermore the production of recombinant AAV5 particlesusing standard packaging systems is approx. 10–50 fold better than AAV2.The majority of the differences in the capsid proteins lies in regionswhich have been proposed to be on the exterior of the surface of theparvovirus. These changes are most likely responsible for the lack ofcross reactive antibodies and altered tissue tropism compared to AAV2.

From the Rep ORF of AAV2, 4 proteins are produced; The p5 promoter (SEQID NO: 18) produces rep 68 (a spliced site mutant) and rep78 and the p19promoter (SEQ ID NO: 16) produces rep 40 (a spliced site mutant) and rep52. While these regions are not well conserved within the Rep ORF ofAAV5 some splice acceptor and donor sites exist in approximately thesame region as the AAV2 sites. These sites can be identified usingstandard computer analysis programs such as signal in the PCGENEprogram. Therefore the sequences of the Rep proteins can be routinelyidentified as in other AAV serotypes.

Hemagglutination Assay

Hemagglutination activity was measured essentially as describedpreviously (Chiorini et al 1997 J. Virol. Vol 71 6823–6833) Briefly 2fold serial dilutions of virus in EDTA-buffered saline were mixed withan equal volume of 0.4% red blood cells in plastic U-bottom 96 wellplates. The reaction was complete after a 2-h incubation at 8° C.Addition of purified AAV5 to a hemagglutination assay resulted inhemagglutination activity.

Transduction of Airway Epithelial Cells

Primary airway epithilial cells were cultured and plated as previouslydescribed (Fasbender et al. J. Clin Invest. 1998 Jul. 1; 102 (1):184-93). Cells were transducted with an equivalent number of rAAV2 orrAAV5 particles containing a nuclear localized β-gal transgene with 50particles of virus/cell (MOI 50) and continued in culture for 10 days.β-gal activity was determined following the procedure of (Chiorini etal. 1995 HGT Vol: δ 1531–1541) and the relative transduction efficiencycompared. As shown in FIG. 7, AAV5 transduced these cells 50-fold moreefficiently than AAV2. This is the first time apical cells or cellsexposed to the air have been shown to be infected by a gene therapyagent.

Transduction of Striated Muscle

Chicken myoblasts were cultured and plated as previously described(Rhodes & Yamada 1995 NAR Vol 23 (12) 2305-13). Cells were allowed tofuse and then transduced with a similar number of particles of rAAV2 orrAAV5 containing a nuclear localized β-gal transgene as previouslydescribed above after 5 days in culture. The cells were stained forβ-gal activity following the procedure of (Chiorini et al. 1995 HGT Vol:6 1531–1541) and the relative transduction efficiency compared. As shownin FIG. 8, AAV5 transduced these cells approximately 16 fold moreefficiently than AAV2.

Transduction of Rat Brain Explants

Primary neonatal rat brain explants were prepared as previouslydescribed (Scortegagna et al. Neurotoxicology. 1997; 18 (2): 331-9).After 7 days in culture, cells were transduced with a similar number ofparticles of rAAV5 containing a nuclear localized β-gal transgene aspreviously described. After 5 days in culture, the cells were stainedfor β-gal activity following the procedure of (Chiorini et al. 1995 HGTVol: 6 1531–1541). As shown in FIG. 9, transduction was detected in avariety of cell types including astrocytes, neuronal cels and glialcells.

Transduction of Human Umbilical Vein Endothelial Cells

Human umbilical vein endothelial cells were cultured and plated aspreviously described (Gnantenko et al. J Investig Med. 1997 February;45(2): 87–98). Cells were transduced with rAAV2 or rAAV5 containing anuclear localized β-gal transgene with 10 particles of virus/cell (MOI5) in minimal media then returned to complete media. After 24 hrs inculture the cells were stained for β-gal activity following theprocedure of Chiorini et al. (1995 HGT Vol: 6 1531–1541), and therelative transduction efficiency compared. As shown in FIG. 10, AAV5transduced these cell 5–10 fold more efficiently than AAV2.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

REFERENCES

-   1. Arella, M., S. Garzon, J. Bergeron, and P. Tijssen. Handbook of    Parvoviruses. Vol. 1. ed. P. Tijssen. Boca Raton, Fla., CRC Press,    1990.-   2. Bachmann, P. A., M. D. Hoggan, E. Kurstak, J. L. MeInick, H. G.    Pereira, P. Tattersall, and C. Vago. 1979. Interverology 11:    248–254.-   3. Bantel-Schaal, U. and M. Stohr. 1992. J. Virol. 66: 773–779.-   4. Chang, L. S., Y. Shi, and T. Shenk. 1989. J. Virol. 63: 3479–88.-   5. Chejanovsky, N. and B. J. Carter. 1989. Virology 173: 120–128.-   6. Chejanovsky, N. and B. J. Carter. 1989. Virology 171: 239–247.-   7. Chiorini, J. A., S. M. Wiener, R. M. Kotin, R. A. Owens, SRM    Kyöstiö, and B. Safer. 1994. J. Virol. 68: 7448–7457.-   8. Chiorini, J. A., M. D. Weitzman, R. A. Owens, E. Urcelay, B.    Safer, and R. M. Kotin. 1994. J. Virol. 68: 797–804.-   9. Chiorini, J. A., C. M. Wendtner, E. Urcelay, B. Safer, M. Hallek,    and R. M. Kotin. 1995. Human Gene Therapy 6: 1531–1541.-   10. Chiorini, J. A., L. Yang, B. Safer, and R. M. Kotin. 1995. J.    Virol. 69: 7334–7338.-   11. Dixit, M., M. S. Webb, W. C. Smart, and S. Ohi. 1991. Gene 104:    253-7.-   12. Fisher, R. E. and H. D. Mayor. 1991. J Theor Biol 149: 429–39.-   13. Flotte, T. R., S. A. Afione, C. Conrad, S. A. McGrath, R.    Solow, H. Oka, P. L. Zeitlin, W. B. Guggino, and B. J. Carter. 1993.    Proc. Natl. Acad. Sci. 90: 10613–10617.-   14. Flotte, T. R., S. A. Afione, R. Solow, M. L. Drumm, D.    Markakis, W. B. Guggino, P. L. Zeitlin, and B. J. Carter. 1993. J    Biol Chem 268: 3781-90.-   15. Hermonat, P. L., M. A. Labow, R. Wright, K. I. Berns, and N.    Muzyczka. 1984. J. Virol. 51: 329–339.-   16. Hermonat, P. L. and N. Muzyczka. 1984. Proc Natl Acad Sci USA    81: 6466-70.-   17. Hunter, L. A. and R. J. Samulski. 1992. J. Virol. 66: 317-24.-   18. Ito, M. and H. D. Mayor. 1968. J. Immuno. 100: 61–68.-   19. Janik, J. E., M. M. Huston, K. Cho, and J. A. Rose. 1989.    Virology 168: 320-9.-   20. Kaplitt, M. G., P. Leone, R. J. Samulski, X. Xiao, D. W.    Pfaff, K. L. O'Malley, and J. M. During. 1994. Nature Genetics 8:    148–154.-   21. Kotin, R. M., M. Siniscalco, R. J. Samulski, X. Zhu, L.    Hunter, C. A. Laughlin, S. McLaughlin, N. Muzyczka, M. Rocchi,    and K. I. Berns. 1990. Proc. Natl. Acad. Sci. (USA) 87: 2211–2215.-   22. Laughlin, C. A., N. Jones, and B. J. Carter. 1982. J. Virol. 41:    868-76.-   23. Laughlin, C. A., M. W. Myers, D. L. Risin, B. J. Carter. 1979.    Virology 94: 162-74.-   24. McCarty, D. M., J. Pereira, I. Zolotukhin, X. Zhou, J. H. Ryan,    and N. Muzyczka. 1994. J. Virol. 68: 4988–4997.-   25. Mendelson, E., J. P. Trempe, and B. J. Carter. 1986. J. Virol.    60: 823–832.-   26. Mizukami, H., N. S. Young, and K. E. Brown. 1996. Virology 217:    124–130.-   27. Muster, C. J., Y. S. Lee, J. E. Newbold, and J. Leis. 1980. J.    Virol. 35: 653-61.-   28. Muzyczka, N. 1992. Curr Top Microbiol Immunol 158: 97–129.-   29. Parks, W. P., J. L. Melnick, R. Rongey, and H. D.    Mayor. 1967. J. Virol. 1: 171–180.-   30. Podsakoff, G., K. K. Jr Wong, and S. Chatterjee. 1994. J. Virol.    68: 5656–5666.-   31. Rose, J. A., M. D. Hoggan, F. Koczot, and A. J.    Shatkin. 1968. J. Virol. 2: 999–1005.-   32. Russell, D. W., A. D. Miller, and I. E. Alexander. 1994. Proc.    Natl. Acad. Sci. USA 91: 8915–8919.-   33. Ryan, J. H., S. Zolotukhin, and N. Muzyczka. 1996. J. Virol. 70:    1542–1553.-   34. Samulski, R. J., K. I. Berns, M. Tan, and N. Muzyczka. 1982.    Proc Natl Acad Sci USA 79: 2077–81.-   35. Samulski, R. J., L. S. Chang, and T. Shenk. 1989. J. Virol. 63:    3822-8.-   36. Sanes, J. R., J. L. R. Rubenstein, and J. F. Nicocas. 1986. EMBO    5: 3133–3142.-   37. Senaphthy, P., J. D. Tratschin, and B. J. Carter. 1984. J Mol    Biol 179: 1–20.-   38. Tratschin, J. D., I. L. Miller, and B. J. Carter. 1984. J.    Virol. 51: 611–619.-   39. Trempe, J. P. and B. J. Carter. 1988. J. Virol. 62: 68–74.-   40. Trempe, J. P., E. Mendelson, and B. J. Carter. 1987. Virology    161: 18–28.-   41. Walsh, C. E., J. M. Liu, X. Xiao, N. S. Young, A. W. Nienhuis,    and R. J. Samulski. 1992. Proc Natl Acad Sci USA 89: 7257-61.-   42. Winocour, E., M. F. Callaham, and E. Huberman. 1988. Virology    167: 393-9.-   43. Jaksch, M., K. D. Gerbitz, and C. Kilger. 1995. Clin. Biochem.    28:503–509-   44. Burcin, M. M., O'Malley, B. W. and S. Y. Tsai. 1998. Frontiers    in Bioscience 3:1–7.

1. A nucleic acid vector comprising an AAV5-specific nucleic acid or anucleic acid encoding an AAV5-specific protein, wherein theAAV5-specific nucleic acid is selected from the group consisting of anAAV5 inverted terminal repeat and an AAV5 p5 promoter, and wherein thenucleic acid encoding an AAV5-specific protein encodes an AAV5-specificprotein selected from the group consisting of AAV5 VP1, AAV5 VP2 andAAV5 VP3, AAV5 capsid, AAV5 Rep40, AAV5 Rep52, AAV5 Rep68 and AAV5Rep78.
 2. A nucleic acid vector of claim 1, comprising a pair ofadeno-associated virus 5 (AAV5) inverted terminal repeats and a promoterbetween the inverted terminal repeats.
 3. The nucleic acid vector ofclaim 2, wherein the promoter is an AAV promoter p5.
 4. The nucleic acidvector of claim 2, wherein the p5 promoter is AAV5 p5 promoter.
 5. Thenucleic acid vector of claim 2, further comprising an exogenous nucleicacid functionally linked to the promoter.
 6. The nucleic acid vector ofclaim 2, encapsidated in an adeno-associated virus particle.
 7. Theparticle of claim 6, wherein the particle is an AAV5 particle.
 8. Theparticle of claim 6, wherein the particle is an AAV1 particle, an AAV2particle, an AAV3 particle, an AAV4 particle or an AAV6 particle.
 9. Anucleic acid vector of claim 1, comprising a nucleic acid encoding anAAV5 capsid protein selected from the group consisting of AAV5 VP1, AAV5VP2, and AAV5 VP3.
 10. A nucleic acid vector of claim 1, comprising anucleic acid encoding an AAV5 Rep protein selected from the groupconsisting of Rep40, Rep52, Rep68 and Rep78.
 11. A nucleic acid vectorof claim 1, comprising a pair of AAV5 inverted terminal repeats.
 12. Thenucleic acid vector of claim 9, wherein the AAV5 capsid protein has theamino acid sequence set forth in SEQ ID NO:
 4. 13. The nucleic acidvector of claim 9, wherein the AAV5 capsid protein has the amino acidsequence set forth in SEQ ID NO:
 5. 14. The nucleic acid vector of claim9, wherein the AAV5 capsid protein has the amino acid sequence set forthin SEQ ID NO:
 6. 15. The nucleic acid vector of claim 10, wherein theAAV5 Rep protein has the amino acid sequence set forth in SEQ ID NO: 2.16. The nucleic acid vector of claim 10, wherein the AAV5 Rep proteinhas the amino acid sequence set forth in SEQ ID NO:
 3. 17. The nucleicacid vector of claim 10, wherein the AAV5 Rep protein has the amino acidsequence set forth in SEQ ID NO:
 12. 18. The nucleic acid vector ofclaim 10, wherein the AAV5 Rep protein has the amino acid sequence setforth in SEQ ID NO:
 14. 19. The nucleic acid vector of claim 11, whereinthe AAV5 inverted terminal repeat has the nucleotide sequence set forthin SEQ ID NO:
 19. 20. The nucleic acid vector of claim 11, wherein theAAV5 inverted terminal repeat has the nucleotide sequence set forth inSEQ ID NO:
 12. 21. A recombinant AAV5 virion containing a vectorcomprising a pair of AAV5 inverted terminal repeats.
 22. The virion ofclaim 21, wherein the vector further comprises an exogenous nucleic acidinserted between the inverted terminal repeats.
 23. An isolated nucleicacid comprising the nucleotide sequence set forth in SEQ ID NO:1.
 24. Anisolated nucleic acid consisting essentially of the nucleotide sequenceset forth in SEQ ID NO:1.
 25. An isolated nucleic acid that selectivelyhybridizes with the nucleic acid of claim 24.